Left ventricular hypertrophy (LVH) and diabetes are among the most potent risk factors for the development of heart failure; furthermore, the combination of diabetes with additional risk factors such as hypertension markedly increases the incidence of heart failure and decreases survival of those diagnosed with heart failure. A key event required for the initiation of hypertrophic signaling is the Ca2+ mediated activation of calcineurin and subsequent nuclear translocation of NFAT, which is currently believed to occur primarily via IP3 mediated Ca2+ release from the SR and nuclear envelope. However, in non-excitable cells, it is widely accepted that it is the subsequent influx of extracellular Ca2+ acros the plasma membrane, so called store operated calcium entry (SOCE) that is essential for activation of calcineurin and NFAT translocation. Recently STIM and Orai protein families have emerged as critical mediators of SOCE in non-excitable cells; however, little is known about the role of these proteins in the heart. The O-linked attachment of ss-N-acetyl-glucosamine (O-GlcNAc) to serine and threonine residues is rapidly emerging as a key mediator of numerous biological processes and has been linked to the adverse effects of diabetes on the heart and also to the regulation of SOCE. We have also recently shown that diabetes impairs cardiomyocyte hypertrophic signaling, at least in part by increased O-GlcNAc levels. Therefore, building on previous reports of SOCE in cardiomyocytes, integrating the recent knowledge of STIM and Orai proteins in mediating voltage- independent Ca2+ entry, combined with our knowledge of protein O-GlcNAcylation, we propose that STIM1-Orai1 facilitated non-voltage gated Ca2+ entry is a key mediator of Ca2+ signaling in adult cardiomyocytes and that O-GlcNAcylation of STIM1 inhibits its normal function thus providing a link between hyperglycemia and abnormal Ca2+-mediated signaling. To test this hypothesis we will pursue 2 specific aims: 1: Demonstrate that STIM1 mediates Ca2+ signaling in adult cardiomyocytes and plays a key role in development of cardiac hypertrophy in vivo; 2: Demonstrate that O-GlcNAc modification of STIM1 inhibits normal activation of STIM1-mediated Ca2+ signaling and contributes to impaired hypertrophic signaling seen in diabetes. We will use gain and loss of function approaches in isolated cardiomyocytes including a novel inducible cardiomyocyte restricted STIM1 knockout mouse to challenge the currently accepted paradigm of Ca2+ homeostasis in adult cardiomyocytes. The successful completion of this proposal will yield significant new insights into the fundamental mechanisms regulating Ca2+ signaling in the heart and establish for the first time a mechanistic link between glucose metabolism and Ca2+ homeostasis and identify novel molecular mediators of cardiac hypertrophy.